Cellobiohydrolase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to cellobiohydrolase variants, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing and using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/081,168, filed Aug. 30, 2018 and published on Mar. 21, 2010 as US2019/0085309 which is a 35 U.S.C. § 371 national application ofPCT/US2017/020502 filed Mar. 2, 2017 and published on Sep. 8, 2017 as WO2017/151957, which claims priority or the benefit under 35 U.S.C. § 119of U.S. Provisional Application No. 62/322,827 filed Apr. 15, 2016 andU.S. Provisional Application No. 62/302,219 filed Mar. 2, 2016, thecontents of which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to cellobiohydrolase variants,polynucleotides encoding the variants, methods of producing thevariants, and methods of using the variants.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars can easily befermented by yeast into ethanol.

WO 2004/016760 discloses variants of Hypocrea jecorina Cel7Acellobiohydrolase I.

WO 2005/001065 discloses variants of Humicola grisea Cel7Acellobiohydrolase I, Hypocrea jecorina cellobiohydrolase I, andScytalidium thermophilium cellobiohydrolase I.

WO 2005/028636 discloses variants of Hypocrea jecorina Cel7Acellobiohydrolase I.

WO 2011/050037 discloses Aspergillus fumigatus cellobiohydrolasevariants with improved thermostability.

WO 2011/050037 discloses Thielavia terrestris cellobiohydrolase variantswith improved thermostability.

WO 2011/123450 discloses Aspergillus fumigatus cellobiohydrolasevariants with improved thermostability.

WO 2012/103288 discloses Talaromyces leycettanus cellobiohydrolasevariants with improved thermostability.

WO 2013/096603 discloses Talaromyces byssochiamydoides cellobiohydrolasevariants with improved thermostability.

U.S. Pat. No. 7,375,197 discloses Trichoderma reesei cellobiohydrolase Ivariants.

The present invention provides cellobiohydrolase variants with improvedproperties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to cellobiohydrolase variants, comprisinga substitution at one or more (e.g., several) positions corresponding topositions 201, 243, 286, and 343 of the polypeptide of SEQ ID NO: 1,wherein the cellobiohydrolase variants have cellobiohydrolase activity.

The present invention also relates to cellobiohydrolase variants,comprising a variant catalytic domain, wherein the variant catalyticdomain comprises a substitution at one or more (e.g., several) positionscorresponding to positions 201, 243, 286, and 343 of SEQ ID NO: 1,wherein the cellobiohydrolase variants have cellobiohydrolase activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition comprising a cellobiohydrolase variant of thepresent invention. In one aspect, the processes further compriserecovering the degraded cellulosic material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a cellobiohydrolasevariant of the present invention; (b) fermenting the saccharifiedcellulosic material with one or more (e.g., several) fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme compositioncomprising a cellobiohydrolase variant of the present invention. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the processes further compriserecovering the fermentation product from the fermentation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show an alignment of a Talaromyces leycettanuscellobiohydrolase (SEQ ID NO: 1), a Trichoderma reesei cellobiohydrolase(SEQ ID NO: 2), a Fusarium solani cellobiohydrolase (SEQ ID NO: 3), aMyceliophthora thermophila cellobiohydrolase (SEQ ID NO: 4), acellobiohydrolase (SEQ ID NO: 5), a cellobiohydrolase (SEQ ID NO: 6), acellobiohydrolase (SEQ ID NO: 7), an Aspergillus fumigatuscellobiohydrolase (SEQ ID NO: 8), and a cellobiohydrolase (SEQ ID NO:9).

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esteraseactivity can be determined using 0.5 mM p-nitrophenylacetate assubstrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esteraseis defined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase.Alpha-L-arabinofuranosidase activity can be determined using 5 mg ofmedium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5in a total volume of 200 μl for 30 minutes at 40° C. followed byarabinose analysis by AMINEX® HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. Alpha-glucuronidase activity can be determined according to deVries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidaseequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Auxiliary Activity 9 polypeptide: The term “Auxiliary Activity 9polypeptide” or “AA9 polypeptide” means a polypeptide classified as alytic polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl.Acad. Sci. USA 108: 15079-15084; Phillips et al., 2011, ACS Chem. Biol.6: 1399-1406; Li et al., 2012, Structure 20: 1051-1061). AA9polypeptides were formerly classified into the glycoside hydrolaseFamily 61 (GH61) according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic material by anenzyme having cellulolytic activity. Cellulolytic enhancing activity canbe determined by measuring the increase in reducing sugars or theincrease of the total of cellobiose and glucose from the hydrolysis of acellulosic material by cellulolytic enzyme under the followingconditions: 1-50 mg of total protein/g of cellulose in pretreated cornstover (PCS), wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of an AA9polypeptide for 1-7 days at a suitable temperature, such as 40° C.-80°C., e.g., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75°C., or 80° C. and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST™ 1.5 L (Novozymes A/S, Bagsværd, Denmark) andbeta-glucosidase as the source of the cellulolytic activity, wherein thebeta-glucosidase is present at a weight of at least 2-5% protein of thecellulase protein loading. In one aspect, the beta-glucosidase is anAspergillus oryzae beta-glucosidase (e.g., recombinantly produced inAspergillus oryzae according to WO 02/095014). In another aspect, thebeta-glucosidase is an Aspergillus fumigatus beta-glucosidase (e.g.,recombinantly produced in Aspergillus oryzae as described in WO02/095014).

AA9 polypeptide enhancing activity can also be determined by incubatingan AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

AA9 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic materialcatalyzed by enzyme having cellulolytic activity by reducing the amountof cellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

The AA9 polypeptide can be used in the presence of a soluble activatingdivalent metal cation according to WO 2008/151043 or WO 2012/122518,e.g., manganese or copper.

The AA9 polypeptide can also be used in the presence of a dioxycompound, a bicyclic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic materialsuch as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO2012/021408, and WO 2012/021410).

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity can be determinedusing 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

Carbohydrate binding module: The term “carbohydrate binding module”means a domain within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose. In an embodiment, the carbohydrate bindingmodule has the sequence of amino acids 19-54 of SEQ ID NO: 1. In anotherembodiment, the carbohydrate binding module has the sequence of aminoacids 25-65 of SEQ ID NO: 2. In another embodiment, the carbohydratebinding module has the sequence of amino acids 25-63 of SEQ ID NO: 3. Inanother embodiment, the carbohydrate binding module has the sequence ofamino acids 26-62 of SEQ ID NO: 4. In another embodiment, thecarbohydrate binding module has the sequence of amino acids 4-39 of SEQID NO: 5. In another embodiment, the carbohydrate binding module has thesequence of amino acids 4-39 of SEQ ID NO: 6. In another embodiment, thecarbohydrate binding module has the sequence of amino acids 2-39 of SEQID NO: 7. In another embodiment, the carbohydrate binding module has thesequence of amino acids 20-57 of SEQ ID NO: 8. In another embodiment,the carbohydrate binding module has the sequence of amino acids 2-40 ofSEQ ID NO: 9.

Catalase: The term “catalase” means ahydrogen-peroxide:hydrogen-peroxide oxidoreductase (E.C. 1.11.1.6 orE.C. 1.11.1.21) that catalyzes the conversion of two hydrogen peroxidesto oxygen and two waters.

Catalase activity can be determined by monitoring the degradation ofhydrogen peroxide at 240 nm based on the following reaction:2H₂O₂→2H₂O+O₂The reaction is conducted in 50 mM phosphate pH 7 at 25° C. with 10.3 mMsubstrate (H₂O₂). Absorbance is monitored spectrophotometrically within16-24 seconds, which should correspond to an absorbance reduction from0.45 to 0.4. One catalase activity unit can be expressed as one μmole ofH₂O₂ degraded per minute at pH 7.0 and 25° C.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme. In anembodiment, the catalytic domain has the sequence of amino acids 107-464of SEQ ID NO: 1. In another embodiment, the catalytic domain has thesequence of amino acids 107-471 of SEQ ID NO: 2. In another embodiment,the catalytic domain has the sequence of amino acids 108-468 of SEQ IDNO: 3. In another embodiment, the catalytic domain has the sequence ofamino acids 119-482 of SEQ ID NO: 4. In another embodiment, thecatalytic domain has the sequence of amino acids 94-451 of SEQ ID NO: 5.In another embodiment, the catalytic domain has the sequence of aminoacids 94-451 of SEQ ID NO: 6. In another embodiment, the catalyticdomain has the sequence of amino acids 94-451 of SEQ ID NO: 7. Inanother embodiment, the catalytic domain has the sequence of amino acids97-454 of SEQ ID NO: 8. In another embodiment, the catalytic domain hasthe sequence of amino acids 90-449 of SEQ ID NO: 9.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo 1 filter paper, microcrystalline cellulose, bacterial cellulose,algal cellulose, cotton, pretreated lignocellulose, etc. The most commontotal cellulolytic activity assay is the filter paper assay usingWhatman No 1 filter paper as the substrate. The assay was established bythe International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in pretreated cornstover (PCS) (or other pretreated cellulosic material) for 3-7 days at asuitable temperature such as 25° C.-80° C., e.g., 25° C., 30° C., 35°C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or80° C., and a suitable pH, such as 3-9, e.g., 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a controlhydrolysis without addition of cellulolytic enzyme protein. Typicalconditions are 1 ml reactions, washed or unwashed PCS, 5% insolublesolids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50° C., 55°C., or 60° C., 72 hours, sugar analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In one aspect, the cellulosicmaterial is any biomass material. In another aspect, the cellulosicmaterial is lignocellulose, which comprises cellulose, hemicellulose,and lignin.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another embodiment, the cellulosic material is arundo, bagasse,bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw, sugarcane straw, switchgrass, or wheat straw.

In another embodiment, the cellulosic material is aspen, eucalyptus,fir, pine, poplar, spruce, or willow.

In another embodiment, the cellulosic material is algal cellulose,bacterial cellulose, cotton linter, filter paper, microcrystallinecellulose (e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another embodiment, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Dissolved Oxygen Saturation Level: The saturation level of oxygen isdetermined at the standard partial pressure (0.21 atmosphere) of oxygen.The saturation level at the standard partial pressure of oxygen isdependent on the temperature and solute concentrations. In an embodimentwhere the temperature during hydrolysis or saccharification is 50° C.,the saturation level would typically be in the range of 5-5.5 mg oxygenper kg slurry, depending on the solute concentrations. Hence, aconcentration of dissolved oxygen of 0.5 to 10% of the saturation levelat 50° C. corresponds to an amount of dissolved oxygen in a range from0.025 ppm (0.5×5/100) to 0.55 ppm (10×5.5/100), such as, e.g., 0.05 to0.165 ppm, and a concentration of dissolved oxygen of 10-70% of thesaturation level at 50° C. corresponds to an amount of dissolved oxygenin a range from 0.50 ppm (10×5/100) to 3.85 ppm (70×5.5/100), such as,e.g., 1 to 2 ppm. In an embodiment, oxygen is added in an amount in therange of 0.5 to 5 ppm, such as 0.5 to 4.5 ppm, 0.5 to 4 ppm, 0.5 to 3.5ppm, 0.5 to 3 ppm, 0.5 to 2.5 ppm, or 0.5 to 2 ppm. In one aspect, thedissolved oxygen concentration during saccharification is in the rangeof 0.5-10% of the saturation level, such as 0.5-7%, such as 0.5-5%, suchas 0.5-4%, such as 0.5-3%, such as 0.5-2%, such as 1-5%, such as 1-4%,such as 1-3%, such as 1-2%.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity can also bedetermined using carboxymethyl cellulose (CMC) as substrate according tothe procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5,40° C.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. Feruloyl esterase activity can be determinedusing 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetatepH 5.0. One unit of feruloyl esterase equals the amount of enzymecapable of releasing 1 μmole of p-nitrophenolate anion per minute at pH5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment hascellobiohydrolase activity. In one aspect, a fragment contains at least380 amino acid residues, at least 400 amino acid residues, or at least420 amino acid residues. In another aspect, a fragment contains at least85% of the amino acid residues, e.g., at least 90% of the amino acidresidues or at least 95% of the amino acid residues of the parentcellobiohydrolase.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 40°C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.,and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, or 9.0.

Hemicellulosic material: The term “hemicellulosic material” means anymaterial comprising hemicelluloses. Hemicelluloses include xylan,glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. Thesepolysaccharides contain many different sugar monomers. Sugar monomers inhemicellulose can include xylose, mannose, galactose, rhamnose, andarabinose. Hemicelluloses contain most of the D-pentose sugars. Xyloseis in most cases the sugar monomer present in the largest amount,although in softwoods mannose can be the most abundant sugar. Xylancontains a backbone of beta-(1-4)-linked xylose residues. Xylans ofterrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.Hemicellulosic material is also known herein as “xylan-containingmaterial”.

Sources for hemicellulosic material are essentially the same as thosefor cellulosic material described herein.

In the processes of the present invention, any material containinghemicellulose may be used. In a preferred aspect, the hemicellulosicmaterial is lignocellulose.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, glucose tolerance,catalytic efficiency, catalytic rate, chemical stability, oxidationstability, pH activity, pH stability, specific activity, stability understorage conditions, substrate binding, substrate cleavage, substratespecificity, substrate stability, surface properties, thermal activity,and thermostability. In particular, the improved property is glucosetolerance, catalytic efficiency, and catalytic rate.

Improved catalytic efficiency: The term “improved catalytic efficiency”means that the ratio of the maximum number of catalytic cycles per unittime an enzyme can carry out under given conditions, such astemperature, pH, dissolved molecules and solute type, divided by theconcentration of substrate required to reach one half of that maximumnumber of catalytic cycles is greater for the variant compared to theparent of the variant.

Improved catalytic rate: The term “improved catalytic rate” means acellobiohydrolase variant converting more substrate to product in agiven period of time compared to the same given amount of the parent ofthe variant in the same period of time under the same conditions, suchas temperature, substrate concentration, substrate composition, pH, saltconcentration, inhibitor concentration.

Improved glucose tolerance: The term “improved glucose tolerance” meansa cellobiohydrolase variant having an improved catalytic rate when mixedwith inhibiting concentrations of glucose compared to the catalytic rateof the parent of the variant in the presence of the same concentrationof glucose and under the same reaction conditions.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Laccase: The term “laccase” means a benzenediol:oxygen oxidoreductase(E.C. 1.10.3.2) that catalyzes the following reaction: 1,2- or1,4-benzenediol+O₂=1,2- or 1,4-benzosemiquinone+2 H₂O.

Laccase activity can be determined by the oxidation of syringaldazine(4,4′-[azinobis(methanylylidene)]bis(2,6-dimethoxyphenol)) to thecorresponding quinone4,4′-[azobis(methanylylidene])bis(2,6-dimethoxycyclohexa-2,5-dien-1-one)by laccase. The reaction (shown below) is detected by an increase inabsorbance at 530 nm.

The reaction is conducted in 23 mM MES pH 5.5 at 30° C. with 19 μMsubstrate (syringaldazine) and 1 g/L polyethylene glycol (PEG) 6000. Thesample is placed in a spectrophotometer and the change in absorbance ismeasured at 530 nm every 15 seconds up to 90 seconds. One laccase unitis the amount of enzyme that catalyzes the conversion of 1 μmolesyringaldazine per minute under the specified analytical conditions.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. It is also known in the art thatdifferent host cells process polypeptides differently, and thus, onehost cell expressing a polynucleotide may produce a different maturepolypeptide (e.g., having a different C-terminal and/or N-terminal aminoacid) as compared to another host cell expressing the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent cellobiohydrolase: The term “parent” or “parentcellobiohydrolase” means a cellobiohydrolase to which an alteration,i.e., a substitution, insertion, and/or deletion, at one or more (e.g.,several) positions, is made to produce the enzyme variants of thepresent invention. The parent may be a naturally occurring (wild-type)polypeptide or a variant or fragment thereof.

Peroxidase: The term “peroxidase” means an enzyme that converts aperoxide, e.g., hydrogen peroxide, to a less oxidative species, e.g.,water. It is understood herein that a peroxidase encompasses aperoxide-decomposing enzyme. The term “peroxide-decomposing enzyme” isdefined herein as a donor:peroxide oxidoreductase (E.C. number 1.11.1.x,wherein x=1-3, 5, 7-19, or 21) that catalyzes the reaction reducedsubstrate (2e⁻)+ROOR′→oxidized substrate+ROH+R′OH; such as horseradishperoxidase that catalyzes the reaction phenol+H₂O₂→quinone+H₂O, andcatalase that catalyzes the reaction H₂O₂+H₂O₂→O₂+2H₂O. In addition tohydrogen peroxide, other peroxides may also be decomposed by theseenzymes.

Peroxidase activity can be determined by measuring the oxidation of2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) by aperoxidase in the presence of hydrogen peroxide as shown below. Thereaction product ABTS_(ox) forms a blue-green color which can bequantified at 418 nm.H₂O₂+2ABTS_(red)+2H⁺→2H₂O+2ABTS_(ox)

The reaction is conducted in 0.1 M phosphate pH 7 at 30° C. with 1.67 mMsubstrate (ABTS), 1.5 g/L TRITON® X-405, 0.88 mM hydrogen peroxide, andapproximately 0.040 units enzyme per ml. The sample is placed in aspectrophotometer and the change in absorbance is measured at 418 nmfrom 15 seconds up to 60 seconds. One peroxidase unit can be expressedas the amount of enzyme required to catalyze the conversion of 1 μmoleof hydrogen peroxide per minute under the specified analyticalconditions.

Pretreated cellulosic or hemicellulosic material: The term “pretreatedcellulosic or hemicellulosic material” means a cellulosic orhemicellulosic material derived from biomass by treatment with heat anddilute sulfuric acid, alkaline pretreatment, neutral pretreatment, orany pretreatment known in the art.

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used area gap open penalty of 10, a gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are a gap open penalty of10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase activity.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the cellobiohydrolaseactivity of the polypeptide of SEQ ID NO: 1.

Wild-type cellobiohydrolase: The term “wild-type” cellobiohydrolasemeans a cellobiohydrolase produced by a naturally occurringmicroorganism, such as a bacterium, yeast, or filamentous fungus foundin nature.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrmann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Reference to “about” a value or parameter herein includes aspects thatare directed to that value or parameter per se. For example, descriptionreferring to “about X” includes the aspect “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that the aspects of the invention describedherein include “consisting” and/or “consisting essentially of” aspects.

Unless defined otherwise or clearly indicated by context, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Conventions for Designation of Variants

For purposes of the present invention, the cellobiohydrolase of SEQ IDNO: 1 is used to determine the corresponding amino acid residue inanother cellobiohydrolase. The amino acid sequence of anothercellobiohydrolase is aligned with the cellobiohydrolase of SEQ ID NO: 1,and based on the alignment, the amino acid position number correspondingto any amino acid residue in the polypeptide of SEQ ID NO: 1 isdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later. The parameters used are a gap open penalty of 10, a gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in anothercellobiohydrolase can be determined by alignment of multiple polypeptidesequences using several computer programs including, but not limited toMUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797); MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When another cellobiohydrolase has diverged from the cellobiohydrolaseof SEQ ID NO: 1 such that traditional sequence-based comparison fails todetect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol.295: 613-615), other pairwise sequence comparison algorithms can beused. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample, the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly, the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated cellobiohydrolase variants,comprising a substitution at one or more (e.g., several) positionscorresponding to positions 201, 243, 286, and 343 of the polypeptide ofSEQ ID NO: 1, wherein the variant has cellobiohydrolase activity.

Variants

The present invention provides cellobiohydrolase variants, comprising asubstitution at one or more (e.g., several) positions corresponding topositions 201, 243, 286, and 343 of SEQ ID NO: 1, wherein the varianthas cellobiohydrolase activity.

In an embodiment, the variant has a sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, to the amino acid sequence of the parentcellobiohydrolase.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 1.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 2.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 3.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 4.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 5.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 6.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 7.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 8.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 9.

The present invention also provides cellobiohydrolase variants,comprising a variant catalytic domain, wherein the variant catalyticdomain comprises a substitution at one or more positions correspondingto positions 201, 243, 286, and 343 of SEQ ID NO: 1, wherein the varianthas cellobiohydrolase activity.

In an embodiment, the variant catalytic domain has a sequence identityof at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, to the catalytic domainof a parent cellobiohydrolase.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 1.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 2.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 3.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 4.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 5.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 6.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 7.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 8.

In another embodiment, the variant catalytic domain has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, such as at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the catalyticdomain of SEQ ID NO: 9.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-4, e.g., 1, 2, 3, or 4 substitutions.

In another aspect, the variant comprises a substitution at one or more(e.g., several) positions corresponding to positions 201, 243, 286, and343. In another aspect, a variant comprises a substitution at twopositions corresponding to any of positions 201, 243, 286, and 343. Inanother aspect, a variant comprises a substitution at three positionscorresponding to any of positions 201, 243, 286, and 343. In anotheraspect, a variant comprises a substitution at each positioncorresponding to positions 201, 243, 286, and 343.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 201. In another aspect, theamino acid at a position corresponding to position 201 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In anotheraspect, the variant comprises or consists of the substitution S201D ofthe polypeptide of SEQ ID NO: 1. In another aspect, the variantcomprises or consists of the substitution S201D of the catalytic domainof SEQ ID NO: 1.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 243. In another aspect, theamino acid at a position corresponding to position 243 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu, Lys, or Val, andmore preferably with Val. In another aspect, the variant comprises orconsists of the substitution L243E, L243K, or L243V of the polypeptideof SEQ ID NO: 1. In another aspect, the variant comprises or consists ofthe substitution L243E, L243K, or L243V of the catalytic domain of SEQID NO: 1.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 286. In another aspect, theamino acid at a position corresponding to position 286 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Ala. In another aspect,the variant comprises or consists of the substitution V286A of thepolypeptide of SEQ ID NO: 1. In another aspect, the variant comprises orconsists of the substitution V286A of the catalytic domain of SEQ ID NO:1.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 343. In another aspect, theamino acid at a position corresponding to position 343 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val, preferably with Glu or Gly. In anotheraspect, the variant comprises or consists of the substitution S343E,G ofthe polypeptide of SEQ ID NO: 1. In another aspect, the variantcomprises or consists of the substitution S343E,G of the catalyticdomain of SEQ ID NO: 1.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201 and 243, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201 and 286, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201 and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 243 and 286, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 243 and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 286 and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201, 243, and 286, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201, 243, and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201, 286, and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 243, 286, and 343, such as thosedescribed above.

In another aspect, the variant comprises or consists of a substitutionat positions corresponding to positions 201, 243, 286, and 343, such asthose described above.

In another aspect, the variant comprises or consists of one or more(e.g., several) substitutions selected from the group consisting ofS201D, L243E,K,V, V286A, and S343E,G at positions in SEQ ID NO: 1 or atpositions corresponding to SEQ ID NO: 1 in other parentcellobiohydrolases, such as those described herein.

In another aspect, the variant comprises or consists of thesubstitutions S201D+L243E,K,V of the polypeptide of SEQ ID NO: 1 or thecatalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+V286A of the polypeptide of SEQ ID NO: 1 or thecatalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+S343E,G of the polypeptide of SEQ ID NO: 1 or thecatalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions L243E,K,V+V286A of the polypeptide of SEQ ID NO: 1 or thecatalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions L243E,K,V+S343E,G of the polypeptide of SEQ ID NO: 1 orthe catalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions V286A+S343E,G of the polypeptide of SEQ ID NO: 1 or thecatalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+L243E,K,V+V286A of the polypeptide of SEQ ID NO: 1or the catalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+L243E,K,V+S343E,G of the polypeptide of SEQ ID NO: 1or the catalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+V286A+S343E,G of the polypeptide of SEQ ID NO: 1 orthe catalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions L243E,K,V+V286A+S343E,G of the polypeptide of SEQ ID NO: 1or the catalytic domain thereof.

In another aspect, the variant comprises or consists of thesubstitutions S201D+L243E,K,V+V286A+S343E,G of the polypeptide of SEQ IDNO: 1 or the catalytic domain thereof.

The variants may further comprise one or more additional alterations,e.g., substitutions, insertions, or deletions at one or more (e.g.,several) other positions.

In one aspect, the variant further comprises a substitution at one ormore (e.g., several) positions corresponding to positions 101, 143, 186,217, 236, 245, 250, 251, 289, 295, 311, 321, 327, 333, 365, 374, 429,and 441 of SEQ ID NO: 1, e.g., E101H, S186Y, A236S, C245L, T251K, N289D,D321N, Q327K, L333F, G365E, G374C, T429Q, and N441C.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

The variants may consist of 400 to 500, e.g., 400 to 450, 410 to 440,415 to 435, and 420 to 440 amino acids.

In an embodiment, the variant has a foreign (heterologous) carbohydratebinding module (a carbohydrate binding module from a different parent).

In an embodiment, the variant has improved catalytic efficiency comparedto the parent enzyme.

In an embodiment, the variant has improved catalytic rate compared tothe parent enzyme.

In an embodiment, the variant has improved glucose tolerance.

Parent Cellobiohydrolases

The parent cellobiohydrolase may be any polypeptide havingcellobiohydrolase activity.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 1, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have cellobiohydrolase activity. In one aspect, the amino acidsequence of the parent differs by up to 10 amino acids, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 1.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 2, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 2.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 3, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 3.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 4, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 4.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 5, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 5.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 6, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 6.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 7, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 7.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 8, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 8.

The parent cellobiohydrolase may be a polypeptide having at least 60%sequence identity to the polypeptide of SEQ ID NO: 9, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the amino acid sequence of the parent differs by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from thepolypeptide of SEQ ID NO: 9.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 1, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have cellobiohydrolase activity. Inone aspect, the amino acid sequence of the catalytic domain of theparent differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, from the catalytic domain of SEQ ID NO: 1.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 2, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 2.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 3, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 3.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 4, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 4.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 5, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 5.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 6, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 6.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 7, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 7.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 8, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 8.

The catalytic domain of a parent cellobiohydrolase may be a catalyticdomain having at least 60% sequence identity to the catalytic domain ofSEQ ID NO: 9, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the amino acid sequence ofthe catalytic domain of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the catalytic domain of SEQID NO: 9.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 1 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 4 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 5 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 6 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 7 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 8 or the catalytic domain thereof.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 9 or the catalytic domain thereof.

In another aspect, the parent is a fragment of the polypeptide of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, or 9 containing at least 380 amino acidresidues, e.g., at least 400 and at least 420 amino acid residues.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be a fungal cellobiohydrolase. For example, the parentmay be a yeast cellobiohydrolase such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacellobiohydrolase; or a filamentous fungal cellobiohydrolase such as anAcremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria cellobiohydrolase.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis cellobiohydrolase.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus lentulus,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillusterreus, Chrysosporium inops, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporiumpannicola, Chrysosporium queenslandicum, Chrysosporium tropicum,Chrysosporium zonatum, Fennellia nivea, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium emersonii, Penicillium funiculosum, Penicilliumpinophilum, Penicillium purpurogenum, Phanerochaete chrysosporium,Talaromyces emersonii, Talaromyces leycettanus, Thermoascus aurantiacus,Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa,Thielavia australeinsis, Thielavia fimeti, Thielavia microspora,Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielaviaspededonium, Thielavia subthermophila, Thielavia terrestris, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cellobiohydrolase.

In another aspect, the parent is a Talaromyces leycettanuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 1.

In another aspect, the parent is a Trichoderma reesei cellobiohydrolase,e.g., the cellobiohydrolase of SEQ ID NO: 2.

In another aspect, the parent is a Fusarium solani cellobiohydrolase,e.g., the cellobiohydrolase of SEQ ID NO: 3.

In another aspect, the parent is a Myceliophthora thermophilacellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 4.

In another aspect, the parent is a cellobiohydrolase of SEQ ID NO: 5.

In another aspect, the parent is a cellobiohydrolase of SEQ ID NO: 6.

In another aspect, the parent is a cellobiohydrolase of SEQ ID NO: 7.

In another aspect, the parent is an Aspergillus fumigatuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 8.

In another aspect, the parent is a cellobiohydrolase of SEQ ID NO: 9.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving cellobiohydrolase activity, comprising: (a) introducing into aparent cellobiohydrolase a substitution at one or more (e.g., several)positions corresponding to positions 201, 243, 286, and 343 of thepolypeptide of SEQ ID NO: 1, wherein the variant has cellobiohydrolaseactivity; and (b) recovering the variant.

In one aspect, the method further comprises introducing a substitutionat one or more (e.g., several) positions corresponding to positions 101,143, 186, 217, 236, 245, 250, 251, 289, 295, 311, 321, 327, 333, 365,374, 429, and 441 of SEQ ID NO: 1, e.g., E101H, S186Y, A236S, C245L,T251K, N289D, D321N, Q327K, L333F, G365E, G374C, T429Q, and N441C.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent. Any site-directed mutagenesisprocedure can be used in the present invention. There are manycommercial kits available that can be used to prepare variants.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Site-saturation mutagenesis systematically replaces a polypeptide codingsequence with sequences encoding all 19 amino acids at one or more(e.g., several) specific positions (Parikh and Matsumura, 2005, J. Mol.Biol. 352: 621-628).

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide recognized by ahost cell for expression of a polynucleotide encoding a variant of thepresent invention. The promoter contains transcriptional controlsequences that mediate the expression of the variant. The promoter maybe any polynucleotide that shows transcriptional activity in the hostcell including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thevariant. Any terminator that is functional in the host cell may be usedin the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thevariant. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillusnigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCI B 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active variant by catalytic or autocatalytic cleavageof the propeptide from the propolypeptide. The propeptide codingsequence may be obtained from the genes for Bacillus subtilis alkalineprotease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of a variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding the variantwould be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising (a) cultivating a recombinant host cell of the presentinvention under conditions conducive for production of the variant; andoptionally (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cells may be cultivated by multi-well plates such as 24, 48, or 96well plates, shake flask cultivation, or small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors in a suitablemedium and under conditions allowing the variant to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variants may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, the whole fermentation broth is recovered.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a variant of the present invention. Thefermentation broth product further comprises additional ingredients usedin the fermentation process, such as, for example, cells (including, thehost cells containing the gene encoding the variant of the presentinvention which are used to produce the variant), cell debris, biomass,fermentation media and/or fermentation products. In some embodiments,the composition is a cell-killed whole broth containing organic acid(s),killed cells and/or cell debris, and culture medium.

The term “fermentation broth” refers to a preparation produced bycellular fermentation that undergoes no or minimal recovery and/orpurification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, a cellulose induced protein (CIP), an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The fermentation broth formulations or cellcompositions may also comprise one or more (e.g., several) enzymesselected from the group consisting of a hydrolase, an isomerase, aligase, a lyase, an oxidoreductase, or a transferase, e.g., analpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of uses of the compositions of the presentinvention. The dosage of the composition and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

Enzyme Compositions

The present invention also relates to compositions comprising a variantof the present invention. Preferably, the compositions are enriched insuch a variant.

The compositions may comprise a variant of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an AA9polypeptide, a CIP, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thecompositions may also comprise one or more (e.g., several) enzymesselected from the group consisting of a hydrolase, an isomerase, aligase, a lyase, an oxidoreductase, or a transferase, e.g., analpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of uses of the compositions of the presentinvention. The dosage of the composition and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the variants having cellobiohydrolase activity, or compositionsthereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition comprising a variant having cellobiohydrolaseactivity of the present invention. In one aspect, the processes furthercomprise recovering the degraded cellulosic material. Soluble productsfrom the degradation of the cellulosic material can be separated frominsoluble cellulosic material using a method known in the art such as,for example, centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a variant havingcellobiohydrolase activity of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme compositioncomprising a variant having cellobiohydrolase activity of the presentinvention. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the processesfurther comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel (ethanol,n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals(e.g., acids, alcohols, ketones, gases, oils, and the like). Theproduction of a desired fermentation product from the cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technology 100: 10-18; Mosier et al., 2005, BioresourceTechnology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on optional addition of achemical catalyst. Residence time for the steam pretreatment ispreferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes,or 4-10 minutes, where the optimal residence time depends on thetemperature and optional addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so that thecellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 2002/0164730). During steam pretreatment, hemicellulose acetylgroups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

Chemical Pretreatment. The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, Bioresource Technology 855: 1-33; Schell et al., 2004,Bioresource Technology 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng.Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, Bioresource Technology 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosicmaterial and held at a temperature in the range of preferably 140-200°C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt.% or 30-60 wt. %, such as around 40 wt. %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment. The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by one ormore enzyme compositions in one or more stages. The hydrolysis can becarried out as a batch process or series of batch processes. Thehydrolysis can be carried out as a fed batch or continuous process, orseries of fed batch or continuous processes, where the cellulosicmaterial is fed gradually to, for example, a hydrolysis solutioncontaining an enzyme composition. In an embodiment the saccharificationis a continuous saccharification in which a cellulosic material and acellulolytic enzyme composition are added at different intervalsthroughout the saccharification and the hydrolysate is removed atdifferent intervals throughout the saccharification. The removal of thehydrolysate may occur prior to, simultaneously with, or after theaddition of the cellulosic material and the cellulolytic enzymecomposition.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s).

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the totalsaccharification time can last up to 200 hours, but is typicallyperformed for preferably about 4 to about 120 hours, e.g., about 12 toabout 96 hours or about 24 to about 72 hours. The temperature is in therange of preferably about 25° C. to about 80° C., e.g., about 30° C. toabout 70° C., about 40° C. to about 60° C., or about 50° C. to about 55°C. The pH is in the range of preferably about 3 to about 9, e.g., about3.5 to about 8, about 4 to about 7, about 4.2 to about 6, or about 4.3to about 5.5.

The dry solids content is in the range of preferably about 5 to about 50wt. %, e.g., about 10 to about 40 wt. % or about 20 to about 30 wt. %.

In one aspect, the saccharification is performed in the presence ofdissolved oxygen at a concentration of at least 0.5% of the saturationlevel.

In an embodiment of the invention the dissolved oxygen concentrationduring saccharification is in the range of at least 0.5% up to 30% ofthe saturation level, such as at least 1% up to 25%, at least 1% up to20%, at least 1% up to 15%, at least 1% up to 10%, at least 1% up to 5%,and at least 1% up to 3% of the saturation level. In a preferredembodiment, the dissolved oxygen concentration is maintained at aconcentration of at least 0.5% up to 30% of the saturation level, suchas at least 1% up to 25%, at least 1% up to 20%, at least 1% up to 15%,at least 1% up to 10%, at least 1% up to 5%, and at least 1% up to 3% ofthe saturation level during at least 25% of the saccharification period,such as at least 50% or at least 75% of the saccharification period.When the enzyme composition comprises an oxidoreductase the dissolvedoxygen concentration may be higher up to 70% of the saturation level.

Oxygen is added to the vessel in order to achieve the desiredconcentration of dissolved oxygen during saccharification. Maintainingthe dissolved oxygen level within a desired range can be accomplished byaeration of the vessel, tank or the like by adding compressed airthrough a diffuser or sparger, or by other known methods of aeration.The aeration rate can be controlled on the basis of feedback from adissolved oxygen sensor placed in the vessel/tank, or the system can runat a constant rate without feedback control. In the case of a hydrolysistrain consisting of a plurality of vessels/tanks connected in series,aeration can be implemented in one or more or all of the vessels/tanks.Oxygen aeration systems are well known in the art. According to theinvention any suitable aeration system may be used. Commercial aerationsystems are designed by, e.g., Chemineer, Derby, England, and build by,e.g., Paul Mueller Company, MO, USA.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, an AA9 polypeptide, a hemicellulase, an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin. In another aspect, the cellulase ispreferably one or more (e.g., several) enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the hemicellulase is preferably oneor more (e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. In another aspect, theoxidoreductase is preferably one or more (e.g., several) enzymesselected from the group consisting of a catalase, a laccase, and aperoxidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anAA9 polypeptide. In another aspect, the enzyme composition comprises anendoglucanase and an AA9 polypeptide. In another aspect, the enzymecomposition comprises a cellobiohydrolase and an AA9 polypeptide. Inanother aspect, the enzyme composition comprises a beta-glucosidase andan AA9 polypeptide. In another aspect, the enzyme composition comprisesan endoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase I, an endoglucanase II, or acombination of an endoglucanase I and an endoglucanase II, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises an endoglucanase I, anendoglucanase II, or a combination of an endoglucanase I and anendoglucanase II, and a beta-glucosidase. In another aspect, the enzymecomposition comprises a beta-glucosidase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase anda cellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, an AA9 polypeptide, and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase I, an endoglucanase II, or a combination of anendoglucanase I and an endoglucanase II, an AA9 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, andan AA9 polypeptide. In another aspect, the enzyme composition comprisesa beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase, anAA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase I, an endoglucanase II, or acombination of an endoglucanase I and an endoglucanase II, abeta-glucosidase, and a cellobiohydrolase I, a cellobiohydrolase II, ora combination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and an AA9 polypeptide. Inanother aspect, the enzyme composition comprises an endoglucanase I, anendoglucanase II, or a combination of an endoglucanase I and anendoglucanase II, a beta-glucosidase, an AA9 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In anembodiment, the xylanase is a Family 10 xylanase. In another embodiment,the xylanase is a Family 11 xylanase. In another aspect, the enzymecomposition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a ligninolytic enzyme. In anembodiment, the ligninolytic enzyme is a manganese peroxidase. Inanother embodiment, the ligninolytic enzyme is a lignin peroxidase. Inanother embodiment, the ligninolytic enzyme is a H₂O₂-producing enzyme.In another aspect, the enzyme composition comprises a pectinase. Inanother aspect, the enzyme composition comprises an oxidoreductase. Inan embodiment, the oxidoreductase is a catalase. In another embodiment,the oxidoreductase is a laccase. In another embodiment, theoxidoreductase is a peroxidase. In another aspect, the enzymecomposition comprises a protease. In another aspect, the enzymecomposition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and/or nativeto the host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and variants having cellobiohydrolaseactivity depend on several factors including, but not limited to, themixture of cellulolytic enzymes and/or hemicellulolytic enzymes, thecellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of a fermenting organism (e.g., for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of the variant havingcellobiohydrolase activity to the cellulosic or hemicellulosic materialis about 0.01 to about 50.0 mg, e.g., about 0.01 to about 40 mg, about0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 toabout 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg,about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per g of thecellulosic or hemicellulosic material.

In another aspect, an effective amount of the variant havingcellobiohydrolase activity to cellulolytic or hemicellulolytic enzyme isabout 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g,about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g ofcellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic or hemicellulosic material, e.g., AA9polypeptides can be derived or obtained from any suitable origin,including, archaeal, bacterial, fungal, yeast, plant, or animal origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtained by,e.g., site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, eachpolypeptide may be a Gram-positive bacterial polypeptide having enzymeactivity, or a Gram-negative bacterial polypeptide having enzymeactivity.

Each polypeptide may also be a fungal polypeptide, e.g., a yeastpolypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides mayalso be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLIC®CTec4 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), SPEZYME™ CP (Genencor Int.), ACCELLERASE™ TRIO(DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W(Röhm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic International, Inc.). Thecellulolytic enzyme preparation is added in an amount effective fromabout 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0wt. % of solids or about 0.005 to about 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase(GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (GenBank:AF487830), Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank:M15665), and Penicillium pinophilumendoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Aspergillus fumigatus cellobiohydrolase I (WO2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (GenBank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

In the processes of the present invention, any AA9 polypeptide can beused as a component of the enzyme composition.

Examples of AA9 polypeptides useful in the processes of the presentinvention include, but are not limited to, AA9 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus(WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascussp. (WO 2011/039319), Penicillium sp. (emersoni0 (WO 2011/041397 and WO2012/000892), Thermoascus crustaceous (WO 2011/041504), Aspergillusaculeatus (WO 2012/030799), Thermomyces lanuginosus (WO 2012/113340, WO2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporusalborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477),Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO2012/135659), Humicola insolens (WO 2012/146171), Malbranchea cinnamomea(WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), Chaetomiumthermophilum (WO 2012/101206), Talaromyces thermophilus (WO 2012/129697and WO 2012/130950), Acrophialophora fusispora (WO 2013/043910), andCorynascus sepedonium (WO 2013/043910).

In one aspect, the AA9 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043 or WO2012/122518, e.g., manganese or copper.

In another aspect, the AA9 polypeptide is used in the presence of adioxy compound, a bicylic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic materialsuch as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO2012/021408, and WO 2012/021410).

In one aspect, such a compound is added at a molar ratio of the compoundto glucosyl units of cellulose of about 10⁻⁶ to about 10, e.g., about10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about10⁻². In another aspect, an effective amount of such a compound is about0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μMto about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M,about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM toabout 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, orabout 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of an AA9 polypeptide can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and an AA9 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485),Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), andTalaromyces thermophilus (GeneSeqP:BAA22816).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum(UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicolainsolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036),Myceliophtera thermophila (WO 2010/014880), Neurospora crassa(UniProt:q7s259), Phaeosphaeria nodorum (UniProt:Q0UHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (UniProt:Q96WX9), Aspergillusterreus (SwissProt:Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

Examples of oxidoreductases useful in the processes of the presentinvention include, but are not limited to, Aspergillus lentiluscatalase, Aspergillus fumigatus catalase, Aspergillus niger catalase,Aspergillus oryzae catalase, Humicola insolens catalase, Neurosporacrassa catalase, Penicillium emersonii catalase, Scytalidiumthermophilum catalase, Talaromyces stipitatus catalase, Thermoascusaurantiacus catalase, Coprinus cinereus laccase, Myceliophthorathermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinuslaccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase,Coprinus cinereus peroxidase, Soy peroxidase, Royal palm peroxidase.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIO-FERM® AFT and XR (Lallemand Specialities, Inc., USA), ETHANOLREDO yeast (Lesaffre et Compagnie, France), FALI® (AB Mauri Food Inc.,USA), FERMIOL® (Rymco International AG, Denmark), GERT STRAND™ (GertStrand AB, Sweden), and SUPERSTART™ and THERMOSACC® fresh yeast(Lallemand Specialities, Inc., USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

In one aspect, the fermenting organism comprises a polynucleotideencoding a polypeptide having cellobiohydrolase activity of the presentinvention.

In another aspect, the fermenting organism comprises one or morepolynucleotides encoding one or more cellulolytic enzymes,hemicellulolytic enzymes, and accessory enzymes described herein.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g., pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term“ketone” encompasses a substance that contains one or more ketonemoieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce the variant inrecoverable quantities. The variant may be recovered from the plant orplant part. Alternatively, the plant or plant part containing thevariant may be used as such for improving the quality of a food or feed,e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed (Sticklen, 2008, Nature Reviews 9: 433-443). For instance,the expression of the gene encoding a variant may be constitutive orinducible, or may be developmental, stage or tissue specific, and thegene product may be targeted to a specific tissue or plant part such asseeds or leaves. Regulatory sequences are, for example, described byTague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, roots,potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet.24: 275-303), or from metabolic sink tissues such as meristems (Ito etal., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter suchas the glutelin, prolamin, globulin, or albumin promoter from rice (Wuet al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from aseed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39:935-941), the storage protein napA promoter from Brassica napus, or anyother seed specific promoter known in the art, e.g., as described in WO91/14772. Furthermore, the promoter may be a leaf specific promoter suchas the rbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiol. 102: 991-1000), the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), thealdP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant,plant part, or a plant cell comprising a polynucleotide encoding thevariant under conditions conducive for production of the variant; andoptionally (b) recovering the variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Media and Solutions

DOB+CSM-Leu plates were composed of 3.4 g of yeast nitrogen base withoutamino acids and ammonium sulfate, 0.68 g of CSM-Leu, 1 ml of 100 mMCuSO₄ 5H₂O, 20 ml of 0.5 M K₂HPO₄, 20 g of Bacto agar, and 950 ml ofdeionized water. Forty ml of a 50% glucose solution were added after theautoclaved medium was tempered to 55° C.

SOC medium was composed of 0.5 g of NaCl, 5 g of yeast extract, 20 g oftryptone, 10 ml of 250 mM KCl, and deionized water to 1 liter.

TBE buffer was composed of 10.8 g of Tris Base, 5 g of boric acid, 4 mlof 0.5 M EDTA pH 8, and deionized water to 1 liter.

TE Buffer was composed of 1 M Tris pH 8.0 and 0.5 M EDTA pH 8.0.

2XYT plates were composed of 16 g of tryptone, 10 g of yeast extract, 5g of NaCl, 15 g of Bacto agar, and deionized water to 1 liter.

2XYT+Amp plates were composed of 2XYT agar supplemented with 100 μg ofampicillin per ml.

YPD medium was composed of 10 g of yeast extract, 20 g of Bacto peptone,40 ml of 50% glucose, and deionized water to 1 liter.

TABLE 1 Primers used in the Examples below Mutagenesis Primers AminoMutant Acid Primer Name Mutation Name Primer Sequence A14-2 L243ETI_CBHII CAGAAGTGTGCTAATGCTCAGAGTGCTTACGAGGAGTGC FwdATCAACTAT (SEQ ID NO: 10) L243E TI_CBHIIGTAAGCACTCTGAGCATTAGCACACTTCTG (SEQ ID NO: 11) Rev L243E V286A TI_CBHIICTTAGCCCGGCCGCTCAACTCTTTGCTTCCGCCTACCAGA Fwd ATGCAAGC (SEQ ID NO: 12)V286A TI_CBHII GGAAGCAAAGAGTTGAGCGGCCGGGCTAAG (SEQ ID NO: 13) Rev V286AS343E TI_CBHII CTGGCTCCATTGCTTCAGCAACAGGGATGGGAGTCAGTT FwdCACTTTATC (SEQ ID NO: 14) S343E TI_CBHIICCATCCCTGTTGCTGAAGCAATGGAGCCAG (SEQ ID NO: 15) Rev S343E A14-4 L243KTI_CBHII CAGAAGTGTGCTAATGCTCAGAGTGCTTACAAGGAGTGC FwdATCAACTAT (SEQ ID NO: 16) L243K TI_CBHIIGTAAGCACTCTGAGCATTAGCACACTTCTG (SEQ ID NO: 17) Rev L243K V286A TI_CBHIICTTAGCCCGGCCGCTCAACTCTTTGCTTCCGCGTACCAG Fwd AATGCAAGC (SEQ ID NO: 18)V286A TI_CBHII GGAAGCAAAGAGTTGAGCGGCCGGGCTAAG (SEQ ID NO: 19) Rev V286AA14-6 L243V TI_CBHII CAGAAGTGTGCTAATGCTCAGAGTGCTTACGTGGAGTGC FwdATCAACTAT (SEQ ID NO: 20) L243V TI_CBHIIGTAAGCACTCTGAGCATTAGCACACTTCTG (SEQ ID NO: 21) Rev L243V Cloning PrimersPrimer Name Primer Sequence 1208418TTGCAGCCAAGATCTCTGCACAGCAAACCATGTGGGGTCA (SEQ ID NO: 22) 1208420TAAATCATATTAATTAAGCTTTAGAAAGAGGGGTTGGCGT (SEQ ID NO: 23) 1209353GCTATTTTTCTAACAAAGCATCTTAGATTA (SEQ ID NO: 24) 1209355GCTGATCCCCTCGTTTTCGGAAACGCTTTG (SEQ ID NO: 25) 1209354GGTCCGTTAAGGTTAGAAGAAGGCTACTTT (SEQ ID NO: 26) 1209356CCTATTCCGAAGTTCCTATTCTCTAGAAAG (SEQ ID NO: 27) 1211892TTGCTATGTACATCGATGCTGGTCATGCTG (SEQ ID NO: 28) 1211893CAGCATGACCAGCATCGATGTACATAGCAA (SEQ ID NO: 29) 1211894GAGCAGAAATACATCAACGCTCTGGCTCCA (SEQ ID NO: 30) 1211895TGGAGCCAGAGCGTTGATGTATTTCTGCTC (SEQ ID NO: 31)

Example 1: Construction of Plasmid pGMER188

Plasmid pGMEr188 is an expression plasmid for the cDNA of the nativeTalaromyces leycettanus CBH II gene. Such cDNA for the T. leycettanusCBH II gene was obtained by designing three 500 bp DNA fragments(G-blocks) with 5′ and 3′ homology with one another to facilitate thecorrect assembly of the gene cDNA. The sequence of the three G-blocksused are listed below and the regions in italics and underlined show theregions of homology between the DNA blocks:

G-block 1: (SEQ ID NO: 32)TGTAAGATCACCCTCTGTGTATTGCACCATGCGGTCTCTCCTGGCTCTTGCCCCTACCCTGCTCGCGCCTGTTGTTCAGGCTCAGCAAACCATGTGGGGTCAATGCGGTGGTCAGGGCTGGACCGGACCTACCATCTGTGTAGCAGGCGCGACATGCAGCACACAGAACCCTTGGTATGCGCAGTGCACCCCAGCACCTACCGCGCCGACGACCTTGCAAACAACAACTACGACGAGCTCGAAATCGTCCACGACCACGAGCTCGAAGTCGTCCACGACCACAGGTGGAAGTGGCGGTGGAACTACGACCTCAACGTCAGCCACCATCACCGCGGCTCCATCTGGTAACCCATACTCCGGATACCAGCTCTATGTGAACCAGGAATACTCGTCCGAGGTGTACGCGTCTGCTATTCCTTCCCTTACCGGCACTCTGGTCGCGAAGGCAAGCGCCGCGGCAGAGGTGCCATCTTTCCTGTGGCTGGACACTGCCTCCAAGG G-block 2: (SEQ ID NO: 33)GAGGTGCCATCTTTCCTGTGGCTGGACACTGCCTCCAAGGTGCCACTGATGGGCACTTACTTGCAGGATATCCAGGCGAAGAACGCTGCTGGCGCCAACCCACCATATGCCGGTCAATTCGTGGTTTACGACTTGCCGGATCGTGATTGCGCTGCATTGGCCAGCAATGGAGAGTACTCCATTGCTAACAATGGTGTTGCCAACTACAAGGCTTACATCGACTCCATCCGCGCGCTTCTTGTTCAATACTCGAACGTCCATGTCATCCTTGTGATCGAGCCCGACAGCTTGGCCAACCTTGTCACCAACCTGAATGTTCAGAAGTGTGCTAATGCTCAGAGTGCTTACCTGGAGTGCATCAACTATGCCCTCACTCAGTTGAACCTCAAGAACGTTGCTATGTACATCGATGCTGGTCATGCTGGATGGCTCGGCTGGCCCGCCAACCTTAGCCCGGCCGCTCAACTCTTTGCTTCCGTATACCAGAATGCAAGCTCCCC G-block 3: (SEQ ID NO: 34)TGCTTCCGTATACCAGAATGCAAGCTCCCCAGCTGCCGTTCGCGGCCTGGCAACCAACGTGGCCAACTATAATGCCTGGTCGATCGCCACTTGCCCATCTTACACCCAAGGCGACCCCAACTGCGACGAGCAGAAATACATCAACGCTCTGGCTCCATTGCTTCAGCAACAGGGATGGTCATCAGTTCACTTTATCACCGATACCGGCCGTAACGGTGTCCAGCCTACCAAGCAGAATGCCTGGGGTGACTGGTGCAACGTTATCGGAACCGGCTTCGGTGTCCGTCCCACCACCAACACTGGCGATCCATTGGAGGATGCTTTCGTCTGGGTCAAGCCTGGTGGTGAGAGTGATGGTACTTCCAACTCCACTTCGCCTCGCTACGACGCCCACTGCGGTTACAGTGATGCTCTTCAGCCTGCTCCTGAGGCTGGTACCTGGTTCGAGGCTTACTTTGAGCAACTCCTTACCAACGCCAACCCCTCTTTCTAATAGTTAA

The sequences highlighted in bold at the beginning of G-block 1 and atthe end of G-block 3 indicate homology with the sense PCR primer 1203966and the antisense PCR primer 1203967 shown below used in the G-blockassembly reaction:

Primer 1203966 (sense): (SEQ ID NO: 35)5′-GCAGCTCACCTGAAGAGGCTTGTAAGATCACCCTCTGTGTATTGCAC C-3′Primer 1203967 (antisense): (SEQ ID NO: 36)5′-CCAACGCCAACCCCTCTTTCTAATAGTTAATTAAGGCTTTCGTGACCG G-3′

The three G-blocks were synthesized by Integrated DNA Technologies, Inc.(Coralville, Iowa, USA), and were received as dry DNA fragments. The 500bp G-block 1, the 500 bp G-block 2 and the 500 bp G-block 3 wereassembled together by PCR using primer 1203966 (sense) and primer1203967 (antisense), resulting in a 1469 bp fragment comprising theentire cDNA for the T. leycettanus CBH II gene. The PCR (50 μl) wascomposed of an equimolecular ratio of the three G-block fragments for atotal amount of DNA of approximately 120 ng, 1× PHUSION® HF buffer(ThermoFisher Scientific), 50 pmol of primer 1203967, 50 pmol of primer1203966, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μl of 100% DMSO,and 1 unit of PHUSION® High Fidelity DNA polymerase (ThermoFisherScientific). The reaction was performed in a thermocycler programmed for1 cycle at 98° C. for 5 minutes; 35 cycles each at 98° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes and 30 seconds;and a final extension cycle at 72° C. for 7 minutes. The completed PCRwas submitted to 0.8% agarose gel electrophoresis in TBE buffer where anapproximately 1469 bp PCR product was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Gel and PCR Clean-up Kit(Macherey-Nagel).

The resulting 1469 bp fragment, comprising the complete T. leycettanusnative CBH II cDNA, was cloned after PCR and gel purification intoplasmid PCR®4-Blunt TOPO® (Life Technologies Corp.) and transformed intoONE SHOT® TOP10 E. coli chemically competent cells (Invitrogen Corp.) byaddition to a single use tube containing the competent cells andincubating the cells on ice for 5 minutes. The tube was incubated at 42°C. for 30 seconds after which 250 μl of SOC medium were added. The tubewas then incubated at 37° C. with agitation at 200 rpm for 1 hour and250 μl were transferred to 2XYT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the desired fragment by sequencing analysis. Onetransformant containing the correct plasmid was identified and theplasmid was designated pGMEr186.

The T. leycettanus CBH II cDNA was amplified by PCR from plasmidpGMEr186 using primer 1203966 (sense) and primer 1203967 (antisense).The PCR (50 μl) was composed of an equimolecular ratio of about 100 ngof plasmid pGMEr186, 1× PHUSION® HF buffer, 50 pmol of primer 1203966,50 pmol of primer 1203967, 200 μM each of dATP, dCTP, dGTP, and dTTP,1.5 μl of 100% DMSO, and 1 unit of PHUSION® High Fidelity DNApolymerase. The reaction was performed in a thermocycler programmed for1 cycle at 98° C. for 5 minutes; 35 cycles each at 98° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for 50 seconds; and a finalextension cycle at 72° C. for 7 minutes. The completed PCR was submittedto 0.8% agarose gel electrophoresis in TBE buffer where an approximately1469 bp PCR product was excised from the gel and purified using aNUCLEOSPIN® Extract II Gel and PCR Clean-up Kit.

The resulting 1469 bp PCR fragment (T. leycettanus CBH II cDNA) wasinserted into Nco I/Pac I-linearized plasmid pJfyS142 (WO 2013/028912)using an IN-FUSION® Advantage PCR Cloning Kit (Clontech Laboratories,Inc.). The reaction was composed of 1× IN-FUSION® Reaction Buffer, 50 ngof Nco I/Pac I-linearized pJfyS142, 100 ng of the T. leycettanus cbh IIcDNA fragment (1469 bp), and 1 μl of IN-FUSION® Enzyme (ClontechLaboratories, Inc.) in a 10 μl reaction volume. The reaction wasincubated for 15 minutes at 50° C. Then 40 μl of TE were added to thereaction and a 2 μl aliquot of the reaction was transformed into ONESHOT® TOP10 E. coli chemically competent cells and transformantsselected as described above. Several of the resulting transformants werescreened for proper insertion of the desired fragment by Sac Irestriction digestion. Plasmid DNA was extracted and purified using aPlasmid Mini Kit (QIAGEN Inc.). A transformant containing a plasmidyielding the desired band sizes of 4223 bp, 3912 bp, 2717 bp, 874 bp and24 bp was isolated and the plasmid was designated pGMEr188.

Example 2: Construction of Yeast Expression Plasmid pLSBF103

Plasmid pLSBF103 was constructed for expression of the Talaromycesleycettanus CBH II (SEQ ID NO: 1) and generation of mutant genelibraries. The Talaromyces leycettanus CBH II cDNA coding sequence (WO2012/103288) was amplified from plasmid pGMER188 (Example 1) using theprimers shown in Table 1. Bold letters represent coding sequence. Theremaining sequences are homologous to insertion sites of pLSBF101.Plasmid pLSBF101 was made by modifying plasmid pDB4081 (WO 2014/072481)to remove the sequence between the promoter and terminator and insert aSaccharomyces cerevisiae invertase leader sequence shown below followedby a Hind III restriction site.

(SEQ ID NO: 37) ATGCTTTTGCAAGCCTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAGATCTCTGCA

Plasmid pLSBF101 was digested with Hind III to linearize the plasmid.The homologous ends of the PCR product and the digested pLSBF101 werejoined together using an IN-FUSION™ Advantage PCR Cloning Kit andtransformed into STELLAR™ competent E. coli cells (ClontechLaboratories, Inc.). Plasmid DNA was purified from transformed coloniesusing a QIAPREP® Spin Miniprep Kit (QIAGEN Inc.). DNA sequencing with a3130XL Genetic Analyzer (Applied Biosystems, Inc.) confirmed thepresence of the CBH II fragment in a final plasmid designated pLSBF103.

Example 3: Construction of Talaromyces leycettanus CBH II Variants

Talaromyces leycettanus CBH II mutant libraries were constructed using atargeted mutagenesis approach. Mutagenic forward primers andcomplementary reverse primers were synthesized for each of the mutationsof interest. Multiple PCR products were used in a yeast-assembly methodto construct each mutant. Using plasmid pLSBF103 (Example 2) as a DNAtemplate, mutations were introduced via PCR using the forward mutagenicprimer for each mutation and a reverse primer downstream of theterminator (SEQ ID NO: 25—Primer 1209355). This reaction results in aPCR product containing a 3′ fragment of the Talaromyces leycettanus CBHII gene containing the mutation of interest, a Saccharomyces cerevisiaealcohol dehydrogenase (ADH1) terminator, and a small amount of DNAnecessary for yeast assembly during the transformation. A second PCR wasperformed using plasmid pLSBF103 as a DNA template with thenon-mutagenic complementary reverse primer for each mutation and aforward primer upstream of the selectable marker (SEQ ID NO: 24—Primer1209353). This reaction results in a PCR product containing a smallamount of DNA necessary for yeast assembly during the transformation, aSaccharomyces cerevisiae 3-isopropylmalate dehydrogenase (LEU2)selectable marker gene, a Saccharomyces cerevisiae protease B (PRB1)promoter, a Saccharomyces cerevisiae invertase leader sequence, and a 5′fragment of the Talaromyces leycettanus CBH II gene. The two productswere then pooled and used in a Splicing by Overlap Extension (SOE) PCRwith a nested primer set (SEQ ID NOs: 26 and 27—Primers 1209354 and1209356) to assemble them into one fragment. When co-transformedalongside linearized plasmid pDB4164 the two DNA fragments come togetherto form a complete 2 micron expression plasmid containing a Talaromycesleycettanus CBH II gene mutant. Plasmid pDB4164 was constructed bymodifying plasmid pDB3936 (WO 2010/092135). It has two additional bases(GC) next to the BamH I site to create a Not I restriction site GCGGCCGC(additional bases in bold) and contains a 1368 bp sequence between theAcc 65I and BamH I sites containing an apramycin resistance selectablemarker.

For variants with multiple mutations a similar procedure was followed.The following steps were used in the construction of mutant A14-4. Inorder to generate the right side fragment a PCR was performed using aV286A CBH 11 mutant as template with the original downstream reverseprimer (SEQ ID NO: 25—Primer 1209355) and a new forward primer justupstream of position 286 (SEQ ID NO: 28—Primer 1211892). A left sidefragment was generated through PCR using a L243K CBH II mutant astemplate with the original upstream forward primer (SEQ ID NO: 24—Primer1209353) and a new reverse primer just downstream of position 243 whichis complementary to the forward primer used in the right side PCR (SEQID NO: 29—Primer 1211893). Following amplification of the right and leftside fragments the two products were pooled and used in a SOE PCR with anested primer set (SEQ ID NOs: 26 and 27—Primers 1209354 and 1209356) toassemble them into one fragment. When co-transformed alongsidelinearized pDB4164 the two DNA fragments came together to form acomplete 2 micron expression plasmid containing a Talaromycesleycettanus CBH II gene with mutations L243K and V286A incorporated.

The following steps were used in the construction of mutant A14-2. Inorder to generate the right side fragment a PCR was performed using aS343E CBH II mutant as template with the original downstream reverseprimer (SEQ ID NO: 25—Primer 1209355) and a new forward primer justupstream of position 343 (SEQ ID NO: 30—Primer 1211894). A left sidefragment was generated through PCR using a CBH II mutant containingL243E and V286A as template with the original upstream forward primer(SEQ ID NO: 24—Primer 1209353) and a new reverse primer just downstreamof position 286 which is complementary to the forward primer used in theRight side PCR (SEQ ID NO: 31—Primer 1211895). Following amplificationof the right and left side fragments the two products were pooled andused in a SOE PCR with a nested primer set (SEQ ID NOs: 26 and27—Primers 1209354 and 1209356) to assemble them into one fragment. Whenco-transformed alongside linearized pDB4164 the two DNA fragments cametogether to form a complete 2 micron expression plasmid containing aTalaromyces leycettanus CBH II gene with mutations L243E, V286A, andS343E incorporated.

Example 4: Transformation and Expression of Variants in Yeast HostStrain

Plasmid pDB4164 DNA was prepared for transformation into S. cerevisiaeas described in WO 2015/036579, Method 4, except that a 9723 bp Acc65I-BamH I fragment from pDB4164 was used as the gapped vector fragmentinstead of the 9721 bp fragment from pDB3936, which has two additionalbases GC next to the BamH I site to create a Not I restriction siteGCGGCCGC. Plasmid pDB4164 also differs from pDB3936 in containing a 1368bp sequence between the Acc 65I and BamH I sites containing an apramycinresistance selectable marker which was excised by the Acc 65I and BamH Idigestion and was not used in the gap-repair transformation. DigestedpDB4164 was co-transformed with a PCR product encoding either wild-typeor mutated Talaromyces leycettanus CBH II. A Saccharomyces cerevisiaestrain (as described in WO 2014/072481) was used as an expression hostfor the Talaromyces leycettanus CBH II variants. This strain was madefrom DYB7 (Payne et al., 2008, Applied and Environmental Microbiology74(24): 7759-7766) with four copies of a protein disulfide isomeraseintegrated into the genome.

Transformed cells were plated onto a selective medium (DOB+CSM-Leu) andallowed to grow at 30° C. for several days. Following the outgrowth, thetransformants were cultured in shake flasks to generate enough materialfor purification. A loop full of cells for each transformant wasre-suspended in 15 μL of culture medium and used as inoculum. The cellswere placed in a 1 L baffled glass shake flask with 200 mL of YPDmedium+100 μM ampicillin and incubated at 30° C. on a 250 rpm shaker for5 days. Following incubation, the cells were pelleted and the brothswere filter sterilized.

Example 5: Purification of Talaromyces leycettanus CBH II VariantsExpressed in Yeast

Each broth (Example 4) was mixed with ½ volume of 20 mM Tris-HCl pH 7.5containing 3.0 M ammonium sulfate to give a final concentration of 1.0 Mammonium sulfate and then filtered using a 0.22 μm polyethersulfonemembrane (Millipore) to remove particulates. Each filtered sample wasapplied to a 75 mL Phenyl Sepharose HP column (GE Healthcare)equilibrated with 1.0 M ammonium sulfate in 20 mM Tris-HCl pH 7.5. Boundproteins were eluted with a decreasing salt gradient (10 column volumes)1.0 M ammonium sulfate to 0 M ammonium sulfate in 20 mM Tris-HCl pH 7.5with 10 mL fractions collected. Fractions were examined by SDS-PAGEusing 8-16% CRITERION™ TGX Stain-Free™ SDS-PAGE gels (Bio-RadLaboratories, Inc.). Each of the variants eluted at ˜400 mM ammoniumsulfate concentration during the gradient. Fractions containing variantwere pooled and were >90% pure as judged by SDS-PAGE. Each pooledmaterial was buffer exchanged into 50 mM sodium acetate pH 5, 100 mMNaCl using four HiPrep™ 26/10 desalting columns (GE Healthcare) linkedin series. Protein concentration was determined by measuring theabsorbance at 280 nm and using the calculated extinction coefficient of2.078 (where a 1 mg/mL solution of the protein would have an absorbanceat 280 nm of 2.078).

Example 6: Characterization of Talaromyces leycettanus GH6 CBH IIVariants PCB+Glc

Cane bagasse was pretreated using low acid steam explosion technique.Prior to enzymatic hydrolysis, the pretreated cane bagasse (PCB) wasground using a COSMOS® Multi Utility Grinder (EssEmm Corporation), andthen was sieved through a 420 micron sieve. The dry content of theground and sieved PCB was adjusted to 6.25%, glucose was added to thesubstrate to a concentration of 50 g/L, and the glucose-enrichedpretreated cane bagasse (PCB) was autoclaved at 121° C. for 30 minutes.Fluorescent brightener 28 (Sigma-Aldrich, CAS #4404-43-7) was added tothe substrate to reach 150 μM in the prepared substrate.

An Enzyme Composition without GH6 Cellobiohydrolase 11

A synthetic enzyme mixture was used as base enzyme, including 49.3%Talaromyces leycettanus GH7 cellobiohydrolase 1, 13.3% Thermoascusaurantiacus GH5 endoglucanase 2, 20% Thermomyces lanuginosus GH61Apolypeptide, 6.7% Trichophaea saccata GH10 xylanase, 6.7% Aspergillusfumigatus beta-glucosidase variant, and 4% Talaromyces emersoniibeta-xylosidase. The enzyme composition is designated herein as“cellulolytic enzyme composition without cellobiohydrolase II”.

All the components used here were at least desalted and buffer-exchangedinto 50 mM sodium acetate pH 5.0 buffer using a HIPREP® 26/10 DesaltingColumn (GE Healthcare), or were further purified through HydrophobicInteraction Chromatography (HIC) or Ion Exchange Chromatography (IEC)(GE Healthcare). The protein concentration was determined using aMicroplate BCA™ Protein Assay Kit (Thermo Fischer Scientific) in whichbovine serum albumin was used as a protein standard.

Hydrolysis of PCB+Glc

Hydrolysis of PCB+Glc (glucose-enriched pretreated cane bagasse (PCB))was performed using the cellulolytic enzyme composition withoutcellobiohydrolase II at 2.0 mg enzyme/g total solids, supplemented by aTalaromyces leycettanus CBH II variant or Talaromyces leycettanuswild-type CBH II at 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg enzyme/gtotal solids, at 55° C., pH 5.0. Total insoluble solids loading of theglucose-enriched PCB was 50 g/L. The glucose concentration was 40 g/L inthe hydrolysis reaction mixture. Total reaction volume was 0.25 ml in96-well plates (Fisher Scientific). Assays were run in triplicate. After72-hour incubation at 55° C., the plates were removed from theincubator, cooled down to room temperature, mixed thoroughly, and readon fluorescence plate reader (Molecular Devices, SpectraMax M5) withbottom read mode, excitation at 365 nm, emittance at 465 nm. The degreeof cellulose conversion was calculated using the following equation, bythe fluorescent readings of:% Conversion=(Sub Ctrl−Reaction)/(Sub Ctrl−Max Digestion)*100%

Several Talaromyces leycettanus CBH II variants showed betterperformance than Talaromyces leycettanus wild-type CBH II at 55° C., pH5.0. Table 2 shows that in the hydrolysis of PCB+Glc, Talaromycesleycettanus CBH II variants A14-2, A14-4 and A14-6, outperformedTalaromyces leycettanus wild type CBH II at 55° C., pH 5.0.

TABLE 2 Conversion by CBH IIs in hydrolysis of PCB + Glc, 55° C., pH5.0. Talaromyces Talaromyces Talaromyces Talaromyces leycettanusleycettanus leycettanus CBH II leycettanus CBH II CBH II CBH II loading,wild-type variant variant variant mg/g TS CBH II A14-2 A14-4 A14-6 0  0%−1%  0%  0% 0.1  9% 10% 10% 10% 0.2 11% 14% 14% 14% 0.3 14% 18% 18% 18%0.4 17% 20% 20% 20% 0.5 19% 23% 21% 21% 0.6 21% 24% 22% 23% 0.7 21% 26%23% 24%

The present invention is further described by the following numberedparagraphs:

[Paragraph 1] A cellobiohydrolase variant, comprising a substitution atone or more positions corresponding to positions 201, 243, 286, and 343of the polypeptide of SEQ ID NO: 1, wherein the variant hascellobiohydrolase activity and wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100%, sequence identity to a parentcellobiohydrolase.

[Paragraph 2] The cellobiohydrolase variant of paragraph 1, wherein theparent cellobiohydrolase has at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[Paragraph 3] The cellobiohydrolase variant of paragraph 1, wherein theparent cellobiohydrolase comprises or consists of SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, or 9.

[Paragraph 4] The cellobiohydrolase variant of paragraph 1, wherein theparent cellobiohydrolase is a fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, or 9, wherein the fragment has cellobiohydrolase activity.

[Paragraph 5] The cellobiohydrolase variant of paragraph 4, wherein thefragment consists of at least 85% of the amino acid residues, e.g., atleast 90% of the amino acid residues or at least 95% of the amino acidresidues of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[Paragraph 6] The cellobiohydrolase variant of any one of paragraphs1-5, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, or 9.

[Paragraph 7] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 1.

[Paragraph 8] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 2.

[Paragraph 9] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 3.

[Paragraph 10] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 4.

[Paragraph 11] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 5.

[Paragraph 12] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 6.

[Paragraph 13] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 7.

[Paragraph 14] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 8.

[Paragraph 15] The cellobiohydrolase variant of any one of paragraphs1-6, which has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the polypeptide of SEQ ID NO: 9.

[Paragraph 16] The cellobiohydrolase variant of any one of paragraphs1-15, wherein the variant consists of 400 to 500, e.g., 400 to 450, 410to 440, 415 to 435, and 420 to 440 amino acids.

[Paragraph 17] A cellobiohydrolase variant, comprising a variantcatalytic domain, wherein the variant catalytic domain comprises asubstitution at one or more positions corresponding to positions 201,243, 286, and 343 of SEQ ID NO: 1 and has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the catalytic domain of a parent cellobiohydrolase.

[Paragraph 18] The cellobiohydrolase variant of paragraph 17, whereinthe catalytic domain of the parent cellobiohydrolase has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the catalytic domain ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[Paragraph 19] The cellobiohydrolase variant of paragraph 17, whereinthe catalytic domain of the parent cellobiohydrolase comprises orconsists of the catalytic domain of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,or 9.

[Paragraph 20] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 1.

[Paragraph 21] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 2.

[Paragraph 22] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 3.

[Paragraph 23] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 4.

[Paragraph 24] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 5.

[Paragraph 25] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 6.

[Paragraph 26] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 7.

[Paragraph 27] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 8.

[Paragraph 28] The cellobiohydrolase variant of paragraph 17, whereinthe variant catalytic domain has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the catalytic domain of SEQ ID NO: 9.

[Paragraph 29] The cellobiohydrolase variant of any one of paragraphs17-28, which further comprises a carbohydrate binding module.

[Paragraph 30] The cellobiohydrolase variant of paragraph 29, whereinthe carbohydrate binding module is a foreign carbohydrate bindingmodule.

[Paragraph 31] The cellobiohydrolase variant of any one of paragraphs1-30, wherein the variant has 1-4, e.g., 1, 2, 3, or 4 substitutions.

[Paragraph 32] The cellobiohydrolase variant of any one of paragraphs1-31, which comprises a substitution at a position corresponding toposition 201 of SEQ ID NO: 1.

[Paragraph 33] The cellobiohydrolase variant of paragraph 32, whereinthe substitution is with Asp.

[Paragraph 34] The cellobiohydrolase variant of any one of paragraphs1-33, which comprises a substitution at a position corresponding toposition 243 of SEQ ID NO: 1.

[Paragraph 35] The cellobiohydrolase variant of paragraph 34, whereinthe substitution is with Glu, Lys, or Val.

[Paragraph 36] The cellobiohydrolase variant of any one of paragraphs1-35, which comprises a substitution at a position corresponding toposition 286 of SEQ ID NO: 1.

[Paragraph 37] The cellobiohydrolase variant of paragraph 36, whereinthe substitution is with Ala.

[Paragraph 38] The cellobiohydrolase variant of any one of paragraphs1-37, which comprises a substitution at a position corresponding toposition 343 of SEQ ID NO: 1.

[Paragraph 39] The cellobiohydrolase variant of paragraph 38, whereinthe substitution is with Glu or Gly.

[Paragraph 40] The cellobiohydrolase variant of any one of paragraphs1-39, which comprises a substitution at two positions corresponding toany of positions 201, 243, 286, and 343 of SEQ ID NO: 1.

[Paragraph 41] The cellobiohydrolase variant of any one of paragraphs1-39, which comprises a substitution at three positions corresponding toany of positions 201, 243, 286, and 343 of SEQ ID NO: 1.

[Paragraph 42] The cellobiohydrolase variant of any one of paragraphs1-39, which comprises a substitution at each position corresponding topositions 201, 243, 286, and 343 of SEQ ID NO: 1.

[Paragraph 43] The cellobiohydrolase variant of any one of paragraphs1-42, which comprises one or more substitutions selected from the groupconsisting of S201D, L243E,K,V, V286A, and S343E,G.

[Paragraph 44] The cellobiohydrolase variant of any one of paragraphs1-43, which further comprises a substitution at one or more positionscorresponding to positions 101, 143, 186, 217, 236, 245, 250, 251, 289,295, 311, 321, 327, 333, 365, 374, 429, and 441 of SEQ ID NO: 1, e.g.,E101H, S186Y, A236S, C245L, T251K, N289D, D321N, Q327K, L333F, G365E,G374C, T429Q, and N441C.

[Paragraph 45] The cellobiohydrolase variant of any one of paragraphs1-44, which has an improved property relative to the parent, wherein theimproved property is selected from the group consisting of improvedglucose tolerance, catalytic efficiency, and catalytic rate.

[Paragraph 46] The cellobiohydrolase variant of any one of paragraphs1-45 which has cellobiohydrolase II activity.

[Paragraph 47] An enzyme composition comprising a cellobiohydrolasevariant of any one of paragraphs 1-46.

[Paragraph 48] The enzyme composition of paragraph 47, furthercomprising one or more enzymes selected from the group consisting of acellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducibleprotein, an esterase, an expansin, a ligninolytic enzyme, anoxidoreductase, a pectinase, a protease, and a swollenin.

[Paragraph 49] The enzyme composition of paragraph 48, wherein thecellulase is one or more enzymes selected from the group consisting ofan endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[Paragraph 50] The enzyme composition of paragraph 48, wherein thehemicellulase is one or more enzymes selected from the group consistingof a xylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[Paragraph 51] The enzyme composition of any one of paragraphs 47-50,further comprising a catalase.

[Paragraph 52] An isolated polynucleotide encoding the cellobiohydrolasevariant of any one of paragraphs 1-46, which is operably linked to oneor more control sequences that direct the production of the polypeptidein an expression host.

[Paragraph 53] A nucleic acid construct comprising the polynucleotide ofparagraph 52.

[Paragraph 54] An expression vector comprising the polynucleotide ofparagraph 52.

[Paragraph 55] A recombinant host cell comprising the polynucleotide ofparagraph 52.

[Paragraph 56] A method of producing a cellobiohydrolase variant,comprising:

(a) cultivating the recombinant host cell of paragraph 55 underconditions conducive for production of the variant; and optionally

(b) recovering the variant.

[Paragraph 57] A transgenic plant, plant part or plant cell transformedwith the polynucleotide of paragraph 52.

[Paragraph 58] A method of producing a cellobiohydrolase variant,comprising:

(a) cultivating a transgenic plant, plant part or a plant cell ofparagraph 57 under conditions conducive for production of the variant;and optionally

(b) recovering the variant.

[Paragraph 59] A method for obtaining a cellobiohydrolase variant,comprising introducing into a parent cellobiohydrolase a substitution atone or more positions corresponding to positions 201, 243, 286, and 343of the polypeptide of SEQ ID NO: 1, wherein the cellobiohydrolasevariant has cellobiohydrolase activity; and recovering the variant.

[Paragraph 60] The method of paragraph 59, further comprisingintroducing into the parent cellobiohydrolase a substitution at one ormore positions corresponding to positions 101, 143, 186, 217, 236, 245,250, 251, 289, 295, 311, 321, 327, 333, 365, 374, 429, and 441 of SEQ IDNO: 1, e.g., E101H, S186Y, A236S, C245L, T251K, N289D, D321N, Q327K,L333F, G365E, G374C, T429Q, and N441C.

[Paragraph 61] A whole broth formulation or cell culture compositioncomprising the cellobiohydrolase variant of any one of paragraphs 1-46.

[Paragraph 62] A process for degrading a cellulosic material,comprising: treating the cellulosic material with an enzyme compositioncomprising the cellobiohydrolase variant of any one of paragraphs 1-46.

[Paragraph 63] The process of paragraph 62, wherein the cellulosicmaterial is pretreated.

[Paragraph 64] The process of paragraph 62 or 63, wherein the enzymecomposition further comprises one or more enzymes selected from thegroup consisting of a cellulase, an AA9 polypeptide, a hemicellulase, acellulose inducible protein, an esterase, an expansin, a ligninolyticenzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.

[Paragraph 65] The process of paragraph 64, wherein the cellulase is oneor more enzymes selected from the group consisting of an endoglucanase,a cellobiohydrolase, and a beta-glucosidase.

[Paragraph 66] The process of paragraph 64, wherein the hemicellulase isone or more enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[Paragraph 67] The process of any one of paragraphs 62-66, furthercomprising recovering the degraded cellulosic material.

[Paragraph 68] The process of paragraph 67, wherein the degradedcellulosic material is a sugar.

[Paragraph 69] The process of paragraph 68, wherein the sugar isselected from the group consisting of glucose, xylose, mannose,galactose, and arabinose.

[Paragraph 70] A process for producing a fermentation product,comprising:

(a) saccharifying a cellulosic material with an enzyme compositioncomprising a cellobiohydrolase variant of any one of paragraphs 1-46;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

[Paragraph 71] The process of paragraph 70, wherein the cellulosicmaterial is pretreated.

[Paragraph 72] The process of paragraph 70 or 71, wherein the enzymecomposition further comprises one or more enzymes selected from thegroup consisting of a cellulase, an AA9 polypeptide, a hemicellulase, acellulose inducible protein, an esterase, an expansin, a ligninolyticenzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.

[Paragraph 73] The process of paragraph 72, wherein the cellulase is oneor more enzymes selected from the group consisting of an endoglucanase,a cellobiohydrolase, and a beta-glucosidase.

[Paragraph 74] The process of paragraph 72, wherein the hemicellulase isone or more enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[Paragraph 75] The process of any one of paragraphs 70-74, wherein steps(a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.

[Paragraph 76] The process of any one of paragraphs 70-75, wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.

[Paragraph 77] A process of fermenting a cellulosic material,comprising: fermenting the cellulosic material with one or morefermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition comprising a cellobiohydrolasevariant of any one of paragraphs 1-46.

[Paragraph 78] The process of paragraph 77, wherein the fermenting ofthe cellulosic material produces a fermentation product.

[Paragraph 79] The process of paragraph 78, further comprisingrecovering the fermentation product from the fermentation.

[Paragraph 80] The process of paragraph 78 or 79, wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.

[Paragraph 81] The process of any one of paragraphs 77-80, wherein thecellulosic material is pretreated before saccharification.

[Paragraph 82] The process of any one of paragraphs 77-81, wherein theenzyme composition further comprises one or more enzymes selected fromthe group consisting of a cellulase, an AA9 polypeptide, ahemicellulase, a cellulose inducible protein, an esterase, an expansin,a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and aswollenin.

[Paragraph 83] The process of paragraph 82, wherein the cellulase is oneor more enzymes selected from the group consisting of an endoglucanase,a cellobiohydrolase, and a beta-glucosidase.

[Paragraph 84] The process of paragraph 82, wherein the hemicellulase isone or more enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[Paragraph 85] The process of any one of paragraphs 62-84, whereinoxygen is added during saccharification to maintain a concentration ofdissolved oxygen in the range of at least 0.5-10% of the saturationlevel.

[Paragraph 86] The method of paragraph 85, wherein the dissolved oxygenconcentration during saccharification is in the range of 0.5-10% of thesaturation level, such as 0.5-7%, such as 0.5-5%, such as 0.5-4%, suchas 0.5-3%, such as 0.5-2%, such as 1-5%, such as 1-4%, such as 1-3%,such as 1-2%.

[Paragraph 87] The process of any one of paragraphs 62-86, wherein theenzyme composition further comprises a catalase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A nucleic acid construct or expression vectorcomprising a polynucleotide encoding a polypeptide variant havingcellobiohydrolase activity, wherein the polynucleotide is operablylinked to one or more heterologous control sequences that direct theproduction of the polypeptide in an expression host, and wherein thepolypeptide variant having cellobiohydrolase activity comprises asubstitution at one or more positions corresponding to positions 201,243, 286, and 343 of the polypeptide of SEQ ID NO: 1, wherein thesubstitution at a position corresponding to position 201 is with Asp,the substitution at a position corresponding to position 243 is withGlu, Lys, or Val, the substitution at a position corresponding toposition 286 is with Ala, and the substitution at a positioncorresponding to position 343 is with Glu or Gly, and wherein thevariant has at least 80%, but less than 100%, sequence identity to SEQID NO: 1, 2, 5, 6, 7, 8, or 9 or at least 95% sequence identity, butless than 100% sequence identity, to SEQ ID NO:
 3. 2. The nucleic acidconstruct or expression vector of claim 1, wherein the polypeptidevariant having cellobiohydrolase activity comprises one or moresubstitutions selected from the group consisting of S201D, L243E,K,V,V286A, and S343E,G of SEQ ID NO:
 1. 3. The nucleic acid construct orexpression vector of claim 1, wherein the polypeptide variant havingcellobiohydrolase activity further comprises a substitution at one ormore positions corresponding to positions 101, 143, 186, 217, 236, 245,250, 251, 289, 295, 311, 321, 327, 333, 365, 374, 421, and 441 of SEQ IDNO:
 1. 4. A recombinant host cell transformed with the nucleic acidconstruct or expression vector of claim
 1. 5. A method of producing acellobiohydrolase variant, comprising: a. cultivating the recombinanthost cell of claim 4 under conditions conducive for production of thevariant; and optionally b. recovering the variant.
 6. A transgenicplant, plant part or plant cell transformed with the nucleic acidconstruct or expression vector of claim
 1. 7. A method of producing acellobiohydrolase variant, comprising: a. cultivating a transgenicplant, plant part or a plant cell of claim 6 under conditions conducivefor production of the variant; and optionally b. recovering the variant.8. A nucleic acid construct or expression vector comprising apolynucleotide encoding a polypeptide variant having cellobiohydrolaseactivity, wherein the poly nucleotide is operably linked to one or moreheterologous control sequences that direct the production of thepolypeptide in an expression host, and wherein the polypeptide varianthaving cellobiohydrolase activity comprises a substitution at one ormore positions corresponding to positions 201, 243, 286, and 343 of thepolypeptide of SEQ ID NO: 1, wherein the substitution at a positioncorresponding to position 201 is with Asp, the substitution at aposition corresponding to position 243 is with Glu, Lys, or Val, thesubstitution at a position corresponding to position 286 is with Ala,and the substitution at a position corresponding to position 343 is withGlu or Gly and wherein the variant has at least 85%, but less than 100%,sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, or
 9. 9. Thenucleic acid construct or expression vector of claim 1, wherein thevariant has at least 96%, but less than 100%, sequence identity to SEQID NO:
 3. 10. The nucleic acid construct or expression vector of claim1, wherein the polypeptide variant having cellobiohydrolase activityfurther comprises a substitution at one or more positions correspondingto positions E101H, S186Y, A236S, C245L, T251K, N289D, D321N, Q327K,L333F, G365E, G374C, T429Q, and N441C of SEQ ID NO: 1.